Klebsiella pneumoniae clinical isolate quickly developed resistance to eravacycline, which is mainly caused by mutations in the gene encoding the Lon protease. The evolved resistant mutants display collateral sensitivities to β-lactam/β-lactamase inhibitor (BLBLI) combinations aztreonam/avibactam and ceftazidime-avibactam. Proteomic analysis revealed upregulation of the multidrug efflux system AcrA-AcrB-TolC and porin proteins OmpA and OmpU, which contributed to the increased resistance to eravacycline and susceptibility to BLBLIs, respectively. The combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam suppresses resistance development. We further demonstrated that eravacycline-resistant mutants evolved from an NDM-1-containing K. pneumoniae strain display collateral sensitivity to aztreonam/avibactam, and the combination of eravacycline with aztreonam/avibactam suppresses resistance development. In addition, the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam displayed synergistic therapeutic effects in a murine cutaneous abscess model. Overall, our results revealed mechanisms of resistance to eravacycline and collateral sensitivities to BLBLIs and provided promising antibiotic combinations in the treatment of multidrug-resistant K. pneumoniae infections.The evolution of bacterial antibiotic resistance is exhausting the list of currently used antibiotics and endangers those in the pipeline. The combination of antibiotics is a promising strategy that may suppress resistance development and/or achieve synergistic therapeutic effects. Eravacycline is a newly approved antibiotic that is effective against a variety of multidrug-resistant (MDR) pathogens. However, the evolution of resistance to eravacycline and strategies to suppress the evolution remain unexplored. Here, we demonstrated that a carbapenem-resistant IMPORTANCE The increasing bacterial antibiotic resistance is a serious threat to global public health, which demands novel antimicrobial medicines and treatment strategies. Eravacycline is a newly approved antibiotic that belongs to the tetracycline antibiotics. Here, we found that a multidrug-resistant Klebsiella pneumoniae clinical isolate rapidly developed resistance to eravacycline and the evolved resistant mutants displayed collateral sensitivity to antibiotics aztreonam/avibactam and ceftazidime-avibactam. We demonstrated that the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam repressed resistance development and improved the treatment efficacies. We also elucidated the mechanisms that contribute to the increased resistance to eravacycline and susceptibility to aztreonam/avibactam and ceftazidime-avibactam. This work demonstrated the mechanisms of antibiotic resistance and collateral sensitivity and provided a new therapeutically option for effective antibiotic combinations. Klebsiella pneumoniae is a Gram-negative opportunistic pathogen that causes various nosocomial infections, such as urinary tract infection, bacteremia, pneumonia, liver abscess, etc. , have been increasingly reported and become a serious public health threat since the early 2000s  (Eravacycline is a novel synthetic fluorocycline that was approved by the USA Food and Drug Administration (FDA) in 2018 for the treatment of complicated intra-abdominal infections . Eravacyes (CRE) . AlthougIn vitro evolution experiments in Acinetobacter baumannii demonstrated that overexpression of the efflux pump genes adeABC results in eravacycline resistance  revealed that Kp43 belonged to ST11, a dominant clone that accounts for up to 60% of carbapenem-resistant K. pneumoniae in China (blaSHV-11 (chromosome), blaOXA-1 (pKp43_1), blaLAP-2 (pKp43_2), blaSHV-12 (pKp43_2), blaCTX-M-65 (pKp43_3), blaKPC-2 (pKp43_3), and blaTEM-1B (pKp43_3). In addition, Kp43 possessed a variety of genes that were involved in resistance to aminoglycoside, quinolone, macrolide, and tetracycline antibiotics . However, Kp43 was susceptible to the BLBLI combinations ceftazidime-avibactam and aztreonam/avibactam , 16K. pnvibactam . Althougceptible , 18.in vitro passage assay with four parallel repeats -PCR assay revealed that the insertion reduced the expression level of lon  mutation , However, expression of the mutated KPC genes in a ceftazidime-avibactam- and aztreonam/avibactam-sensitive strain PA4325 did not increase the resistance to ceftazidime-avibactam or aztreonam/avibactam, indicating that the mutations were not responsible for the resistance . The MICs of eravacycline for the ceftazidime-avibactam-resistant strains decreased 2-fold, indicating collateral sensitivity. However, no change was observed in the aztreonam/avibactam-resistant mutants , whereas no mutation was found in the lon genes of the strains of Kp17-C1-C3. Because Kp17 was resistant to multiple antibiotics used in cloning, including apramycin, we were unable to complement the mutants by introducing a wild-type lon gene into the evolved strains.To examine whether the collateral sensitivities and the resistance development suppressive effects of the combinations were reproducible on another clinical isolate, we performed a passage assay with a New Delhi metallo-beta-lactamase (NDM)-1-containing We next examined the collateral sensitivity of the three evolved eravacycline-resistant mutants. The parental strain Kp17 was highly resistant to ceftazidime-avibactam because avibactam is unable to inhibit NDM-1 . HoweverIn addition to suppression of resistance development, another important objective of combination therapy is to improve antimicrobial efficacies . We, thuin vivo bactericidal activities of the combinations. The dose of each drug was used following previous reports  and the European Medicines Agency (EMA) in 2015 and 2016, respectively. However, ceftazidime-avibactam-resistant ereafter –49. In v isolate , 51. In  isolate –51. For ztreonam , 53. In ztreonam , 53.K. pneumoniae clinical isolate (Kp17) displayed reciprocal collateral sensitivity between eravacycline and aztreonam/avibactam. The combination of eravacycline and aztreonam/avibactam also suppressed the development of resistance in Kp17. Further studies are warranted to understand the mechanism that leads to the difference in the collateral sensitivities to aztreonam/avibactam of the eravacycline-resistant mutant evolved from Kp43 and Kp17.Here, we found that the evolved eravacycline-resistant mutants of Kp43 displayed collateral sensitivity to ceftazidime-avibactam and aztreonam/avibactam. Meanwhile, the evolved ceftazidime-avibactam-resistant strains were more susceptible to eravacycline. However, the evolved aztreonam/avibactam-resistant strains maintained the same level of resistance to eravacycline. The combination of eravacycline with ceftazidime-avibactam or aztreonam/avibactam suppressed the development of resistance to each of the antibiotics and their combinations. An NDM-1-containing K. pneumoniae effects in vitro and synergistic bactericidal effects in vivo. Transcriptomic and proteomic studies are needed to understand the bacterial response to the combinations and elucidate the mechanisms of the synergy.The eravacycline-ceftazidime-avibactam and eravacycline-aztreonam/avibactam combinations displayed close to synergy anti-Eravacycline has been approved for the treatment of complicated intra-abdominal infections (cIAIs) in several countries , 32. In The doses of eravacycline, ceftazidime-avibactam, and aztreonam/avibactam for adults are around 4, 29/7.25, and 29/7.25 mg/kg, respectively , 32, 55.in vivo free zinc concentration is lower than that in MHB  NDM-1, whose activity depends on zinc. However, the t in MHB , 57. A rK. pneumoniae clinical isolates Kp43 and Kp17 were isolated from blood and sputum culture samples from two patients in Beijing, China. Gene deletion and complementation were performed as previously described . The PCR product was cloned into the XbaI-NheI sites of the plasmid pKP, resulting in pKP-lonKp43 . The plasmid was transferred into the lon mutants by electroporation.For complementation of the MICs were determined by the 2-fold serial dilution method in cation-adjusted Mueller-Hinton broth (CAMHB) following the Clinical and Laboratory Standards Institute (CLSI) . Avibact5 CFU/mL in a 96-well microliter plate. After incubation at 37°C for 20 h, the MIC values were defined as the lowest concentrations of antibiotics with no visible growth of bacteria. FICIs were calculated using the formula: FICI =  + . The results were interpreted according to EUCAST  of 2.0 were inoculated into fresh CAMHB with increased concentrations of eravacycline  for another round of passage. The passaging was repeated for 8 days, and the replicates were streaked to obtain single colonies for MIC determination.The strains Kp43 and Kp17 were passaged in the presence or absence of eravacycline. Overnight culture of the bacteria was subcultured into fresh CAMHB with increasing concentrations of eravacycline . After 24 h, cells from the highest concentration of antibiotic that allowed growth to a minimum optical density at 600 nm  . MICs of the combinations were determined by mixing eravacycline with aztreonam or ceftazidime at a 1:1 ratio in the presence of 4 mg/L avibactam. After 24 h, cells from the highest concentration of antibiotic that allowed the growth to a minimum OD600 of 2.0 were inoculated into freshly CAMHB with increasing concentrations of the antibiotics for another round of passage. The passaging was repeated for 8 days, and the replicates were streaked to obtain single colonies for MIC determination.To examine the development of resistance to antibiotic combinations, overnight cultures of the Sequencing of the Kp43 genome was performed by GrandOmics  by Nanopore sequencing using the PromethION platform. Genomes of the four eravacycline-resistant mutant colonies Kp43-E1, Kp43-E2, Kp43-E3, and Kp43-E4 and one control strain passaged in CAMHB (designated Kp43-C1) were sequenced using the MGISEQ2000 platform. Paired-ended sequenced raw reads were filtered using the fastp (v.0.20.0) preprocessor, and then the clean reads were mapped to the reference genome of the parental strain Kp43 using bwa software. Freebayes, snpEff, and bcftools were used for variant calling of single nucleotide polymorphisms (SNPs), small insertions, and deletions (indels).600 of 1.0. Total RNA was extracted and reverse transcribed to cDNA. The cDNA was mixed with the indicated primers  and SYBR premix Ex Taq II . Then, qRT–PCR was performed in a Step One Plus system . The DNA-directed RNA polymerase beta chain gene rpoB was used as an internal control.Bacterial overnight cultures were inoculated into fresh LB medium and grown to an OD600 of 1 in LB. One milliliter of the bacteria was collected by centrifugation and resuspended in 1 mL LB, followed by incubation with 50 mg/L eravacycline for 15 min. The bacteria were collected by centrifugation and washed with 1 mL of an Mg2+ buffer . The bacteria were then resuspended in 1 mL of the Mg2+ buffer. A 200 μL bacterial suspension was added to each well of a 96-well plate and the fluorescence (excitation at 400 nm and emission at 520 nm) was immediately measured with a Varioskan Flash reader . For pretreatment with the efflux pump inhibitor PAβN, the bacteria were incubated with 50 mg/L PAβN for 1 h in LB before incubation with eravacycline.Eravacycline accumulation was determined as previously described with minor modifications . Kp43 an600 of 0.8 in LB, followed by incubation with 50 mg/L PAβN for 1 h. The cells were washed once and resuspended in the same volume of PBS. Then, half of the cells were lysed by sonication, and the supernatant was collected after centrifugation. The intact cells or the supernatant were incubated with 64 mg/L ceftazidime at room temperature. The OD260 was measured with a Varioskan Flash reader  after 1 h in a 96-well plate.The ceftazidime influx assay was performed as previously described with modifications . Bacteri600 of 1. The bacteria were mixed with 10 mM dithiothreitol (DTT), followed by a water bath at 56°C for 1 h. The samples were cooled, mixed with 55 mM iodoacetamide (IAA), and incubated at room temperature in the dark for 45 min. Then, cold acetone was added to the solution and incubated at −20°C for 30 min. Protein samples were collected by centrifugation at 25,000 g at 4°C for 15 min and air dried. The pellets were resolubilized in buffer , and the protein concentrations were determined by bovine serum albumin (BSA) tryptophan assay. The samples were diluted with 0.5 M triethylammonium bicarbonate (TEAB) and digested with a mixture of trypsin enzyme  at 37°C for 4 h. Then, the digested peptides were desalted and freeze-dried.Bacterial membrane proteins were extracted using a bacterial membrane protein extraction kit . Briefly, bacteria were grown in LB to ODThe LC-MS/MS assays were performed by the Beijing Genomics Institute, Beijing, China. The dried peptide samples were reconstituted with mobile phase A  and then centrifuged at 20,000 g for 10 min. The supernatant was taken for injection. Separation was performed by a SCIEX eksigent ultra 2D model nanoliter liquid chromatography. The liquid-phase chromatography-separated peptides were passed to an ESI tandem mass spectrometer.The animal experiments were conducted following the national guidelines on the use of animals in research. The protocol was approved by the Animal Care and Use Committee of Nankai University, College of Life Sciences (permission number: NK-04-2392012).600 of 1 in LB, washed twice with sterile saline, and resuspended at 4 × 108 CFU/mL. 50 μL of the bacteria were injected into the right side of the dorsum, resulting in 2 × 107 CFU per mouse. Antibiotics or saline (50 μL) was injected subcutaneously into the infected area at 45 min postinfection. After 20 h, the skin abscesses  were excised and homogenized in sterile saline. The bacterial counts were determined by 10-fold serial dilution with saline and plating.The mouse cutaneous abscess infection was performed as previously described  with minSRR18724480). The WGS data of the evolved Kp43 mutants were also deposited in the NCBI database (SRR18748512 to SRR18748516). The protein sequences of Kp43 were included in Supplemental File 2.The genome sequence of Kp43 was deposited in the NCBI database (


Management of potentially malignant oral disorders pose a challenge to the clinician. These lesions, over time, can undergo cellular and morphologic alteration (dysplasia) until they evolve into squamous cell carcinoma. Diagnosis is made by biopsy and histologic analysis. Screening methods that noninvasively allow these lesions to be monitored both diagnostically and prognostically (biomarkers) have long been sought. A class of non-coding RNAs called miRNAs is readily found in biological fluids and in saliva: they fit into this area of research. From the data presented in this study, miR-181 appears to be a promising salivary biomarker for dysplasia.p = 0.046); miR-181b was upregulated in high-grade dysplasia (p = 0.006), increasing with the degree of dysplasia, and decreasing in OSCCs. (4) Conclusions: Salivary miR-27b and miR-181b could be promising biomarkers for oral dysplasia. Further studies are needed to clarify their feasibility.(1) Background: Oral potentially malignant disorders (OPMD) represent a fundamental challenge for clinicians, considering the possibility of progression into oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Several studies have examined the expression of miRNAs in humans as diagnostic and prognostic biomarkers. Among these, miR-21, miR-27b, and miR-181b proved to be promising. This cohort study evaluated the different expressions of those miRNAs in the saliva of patients with OPMD and OSCC. (2) Methods: Patients with a clinical diagnosis of OPMD and/or OSCC were enrolled; saliva samples were collected; miRNAs were extracted and quantified via qRT-PCR was performed. Data were analyzed by subgroups based on the histopathological diagnosis (OSCC and the grade of OED) using the ΔΔCt method. Saliva from 10 healthy donors was used as the control. One-way ANOVA and Kruskal–Wallis tests were performed to assess the differences between groups. (3) Results: 23 patients for the OPMD group  and 10 with OSCC were analyzed. MiR-21 did not show any variation among groups; miR-27b was under-expressed in dysplastic lesions ( Saliva represents a biological fluid rich in electrolytes, hormones, proteins, peptides, and nucleic acids, including non-coding RNAs . These nSeveral studies have shown that miRNAs in saliva, wrapped up by proteins or stored within exosomes, are stable and can resist degradation. MiRNAs could enter saliva from the blood through various cellular mechanisms, such as transcellular or paracellular ways, or they could also be produced locally by cellular apoptosis or necrosis and could also be released from normal epithelial or cancer cells in exosomes. Profiling of miRNAs isolated from exosomes and their donor cells indicates that exosomes are highly enriched in RNA, as well as miRNAs, which reflect their cellular expression ,7.In contrast, miRNA dysregulation may be associated with the pathogenesis of inflammatory and malignant diseases . RepressOSCC is a type of cancer characterized by a low survival rate ,20. ThisFor this cohort study, the manuscript was prepared and revised according to the STROBE Statement–checklist of items .Thirty-six patients were recruited consecutively (from May to December 2021) from the Oral Medicine Division and Internal Medicine Units of the University of Campania “Luigi Vanvitelli.” For the study group, patients of both sexes aged over 18 with a clinical diagnosis of OPMD and/or OSCC have been enrolled. Ten healthy patients of both sexes aged over 18 were enrolled in the control group .In both groups, patients with other malignancies (non-OSCC) and/or under treatment with chemo and radiotherapy, patients who have undergone an organ transplant, patients with a positive history of chronic viral diseases, and patients under therapy with systemic or topical oral corticosteroids were excluded. Patients with drug-induced or contact lichenoid reactions and pregnant or lactating women were also excluded. Moreover, all patients enrolled were tested for SARS-CoV-19 infection before sample collection, and we selected only SARS-CoV-19-free patients.Cigarette and alcohol consumption were not considered among the exclusion criteria. However, patients were asked to suspend for at least one week before being able to do the salivary samples, as well as for antibiotics, NS anti-inflammatory agents, analgesics, or chlorhexidine mouthwashes. The same was required of the participants of the control group.At the examination, the salivary samples were collected. All patients of the study group underwent histological analysis; they were subsequently divided into the following subgroups based on their histopathological diagnosis  : (a) OSCWritten informed consent was obtained from all participants. The study was approved by the ethical committee of the University of Campania “Luigi Vanvitelli” (#0000339/2018). Details regarding patients of all groups are reported in The protocol for collecting all saliva samples was developed according to the standards presented in the literature . The sal® MicroRNA Reverse Transcription Kit (Thermofisher Scientific), single-stranded cDNA was synthesized from total RNA samples, as reported in our previous work [® PreAmp-Master Mix using sequence-specific primers and probes on the Megaplex PreAmp primers. The pre-amplified products were used to evaluate the different expressions of miR-21, miR-27b, and miR-181b by quantitative real-time PCR (qRT-PCR)  using TaqMan miRNA Assays that use looped-primer to detect mature miRNAs accurately. qRT-PCR was performed on a ViiA7 Real-time PCR system . The small-nuclear-U6 was selected as an appropriate constitutively expressed endogenous control to normalize total RNA samples. The transcripts’ relative expression was measured using ViiA7-Real-Time PCR software .The MiRNA extraction was performed using a MIRVANA PARIS KIT . It was optimal for our purpose because of its higher sensitivity. Subsequently, using the TaqManous work . Then, cThe study’s primary outcome is to evaluate the expression of miRNAs miR-21, miR-27b, and miR-181b in subjects with OPMD and OSCC by comparing the data obtained with a control group. Specifically, the following analyses were performed: (1) miRNA expression in the OPMD group; (2) miRNA expression in the OSCC group; (3) OSCC group vs. OPMD group. The secondary outcome aims to analyze, within the OPMD group, the expression of selected miRNAs according to the degree of dysplasia (binary system): high-grade (HG) dysplasia vs. low-grade (LG) dysplasia and no dysplasia (ND).® SPSS® Statistics (ver. 28.0.1.0).Statistical analysis within the study subjects was carried out and compared with the control group. Analyses were performed using IBMIn order to assess the statistical power and significance of ΔCt variation in OPMD, OSCC and control groups, the power analysis was performed using G*Power 3.1.9.6  with a one-way ANOVA assuming a medium Cohen’s effect size of 0.5 (α = 0.05). Sensibility  was fixed at 0.80.The expression of each miRNA tested was normalized by the ΔΔCt method . The 2-Δ® SPSS® Statistics (ver. 28.0.1.0.) was created, and ΔCt difference among groups was calculated using one-way ANOVA. Post hoc tests were used for multiple comparisons. The Kruskal–Wallis test was used to assess the differences within the OPMD group; the Mann–Whitney test was used to test the expression of miRNAs depending on the OSCC grade. The Spearman test was performed to exclude correlation between the independent variables. A receiver operating characteristic (ROC) curve was made to assess the diagnostic ability of the analyzed miRNAs for OED and OSCC. The significance level was considered at p ≤ 0.05. When required, a Bonferroni correction has been applied. Graphs were produced with GraphPad Prism (ver. 9.4.1-458).A database system using IBMA total of 46 patients with a mean age of 60.02 ± 16.22 years  were enrolled in the study. Three patients (2 with OPMD and 1 with OSCC) were excluded from the study because the quality of the collected salivary samples did not allow analysis. The OPMD group consisted of 23 people  with a mean age of 61.22 ± 11.87 years; among these, six patients presented epithelial hyperkeratosis with no dysplasia (ND), seven were diagnosed with low-gradeOED (LG), and 10 with high-grade OED (HG).p > 0.05).Ten patients were enrolled in the OSCC group  with a mean age of 69.90 ± 9.78. Seventy percent of the OSCCs were localized to the tongue (four ulcerated and three nodular), 20% were exophytic lesions at the level of the alveolar ridge, and finally, one ulcerated lesion at the level of the retromolar trigone. Histopathological analysis established the grading of the lesions: 50% were in G1, and 50% were in G2. The expression of miR-21, miR-27b, and miR-181b did not show a significant difference between G1 and G2 lesions (p = 0.523), miR-27b (p = 0.691), and miR-181b (p = 0.389) was not found in the present study.The control group, consisting of 10 healthy volunteers , had a mean age of 45.20 ± 20.22 years. The age of patients in the control group is significantly lower than in the other groups; however, a correlation between age and expression of miR-21  has been found, while miR-27b reported a variation between groups (p = 0.046). The difference between its concentration in patients with dysplastic and non-dysplastic lesions was not significant (p = 0.77). However, it was under-expressed in dysplastic lesions by 4.52 folds (p = 0.042) compared with controls. The salivary concentration of miR-181b varied among the groups (p < 0.001). In particular, there was overexpression in patients with dysplasia compared with controls of 5.14 folds (p = 0.006) and non-dysplastic lesions of 6.29 folds (p = 0.014). There was no difference in miR-181b expression between controls and non-dysplastic lesions (p = 0.717)—p < 0.001 (p = 0.66).No variation in the salivary expression of miR-21 ( < 0.001 . At the p = 0.062). The salivary concentration of miR-27b changed in the groups (p = 0.018), but there was no difference between OSCC and non-dysplastic lesions (p = 0.863), dysplastic lesions (p = 0.051) and control group (p = 0.905). MiR-181b was under-expressed in patients with OSCC of 7.39 folds (p < 0.001) compared to patients with dysplastic lesions. However, the expression of this miRNA was not significant when comparing non-dysplastic lesions (p = 0.891) and controls (p = 0.253).The expression of miR-21 did not change in the different groups (p = 0.197) showed no significant differences (p = 0.032), but multiple comparisons showed no significant differences . Moreover, expression of salivary miR-181b increased with increasing degree of dysplasia, discriminating between HG dysplasia and LG dysplasia (2.6 folds difference—p = 0.04) and OPMD without dysplasia (7.27 folds difference—p< 0.001) as shown in Salivary concentrations of miR-181b appeared to vary significantly compared with controls. Specifically, in patients with HG dysplasia, miR-181b expression was upregulated, compared with controls, by about 6.1-fold (p = 0.509) and non-dysplastic lesions (p = 0.948). At the same time, it is under-expressed concerning HG dysplasia (8.37 folds difference—p < 0.001) and LG dysplasia, as reported in p < 0.001).The expression of this miRNA in OSCC does not differ from controls  could coincide with an increased expression of this miRNA in the saliva. In an ex vivo study of the expression of specific miRNAs in dysplastic samples of oral leukoplakia, Brito et al. found increased expression of miR-181b in samples with a higher number of mitotic figures, increased nuclear/cytoplasmic ratio, or hyperchromia . MoreoveRegarding miR-27b, data from the present study show an under-expression of this miRNA in dysplastic lesions compared with controls but no difference between this group and the OSCC group. In previous work, this miRNA was found to be down-expressed in the saliva of patients with OLP (which is an OPMD) without dysplasia . MoreoveFinally, miR-21 is one of the miRNAs most associated with the mechanisms of malignant transformation of oral lesions. A recent systematic review indicated that miR-21 could provide a prognostic indicator in OSCC . The datThe main limitation of this pilot study is the sample size. For this type of salivary sampling study and subsequent validation of a biomarker for dysplasia and OSCC, multicenter studies with a larger population will be necessary. In addition, it would be interesting to follow the evolution of the dysplastic lesions’ development at different time points, evaluate the expression of these miRNAs in the eventual malignant transformation at the same oral site, and compare the expression of salivary miRNAs with those on biopsy tissue.In conclusion, increasing data shows that miRNAs play an important role in cancer pathogenesis and OED progression ,21. Alth


This study provides scientific support for the preparation of f-Zn-modified epoxy resin.As a typical brittle material, epoxy resin cannot meet its application requirements in specific fields by only considering a single toughening method. In this paper, the effects of carboxyl-terminated polybutylene adipate (CTPBA) and zinc powder on the mechanical properties, adhesion properties, thermodynamic properties and medium resistance of epoxy resin were studied. A silane coupling agent (KH-550) was used to modify zinc powder. It was found that KH-550 could significantly improve the mechanical properties and bonding properties of epoxy resin, and the modification effect of flake zinc powder (f-Zn) was significantly better than that of spherical zinc powder (s-Zn). When the addition amount of f-Zn was 5 phr, the tensile shear strength and peel strength of the composites reached a maximum value of 13.16 MPa and 0.124 kN/m, respectively, which were 15.95% and 55% higher than those without filler. The tensile strength and impact strength reached a maximum value of 43.09 MPa and 7.09 kJ/m Epoxy resin has the advantages of excellent mechanical properties, a high bonding strength, low curing shrinkage, good corrosion resistance and a low price. It is widely used in the preparation of adhesives, composite materials and coatings ,2,3. HowThe common methods of toughening modifications of epoxy resin include adding rubber elastomer, thermoplastic resin, inorganic nanoparticles and flexible segments ,10,11. RAs a typical brittle material, epoxy resin cannot meet the application requirements of specific fields by only considering a single toughening method. The synergistic modification of epoxy resin can achieve the purpose of toughening and thermal performance improvement ,20,21. IPolymer composites not only inherit a series of advantages from polymers, but also have the advantages of a reinforcing phase, which have been widely used in the flame retardant, anticorrosion and electromagnetic fields ,24,25,26In this paper, bisphenol A epoxy resin (E-51) was used as the research object, phenolic amine (T-31) was used as the curing agent, epoxy-terminated polybutylene adipate (E-CTPBA) was used as the toughening agent  and zin3, ≥99.0%), an analytical reagent, was purchased from Aladdin Reagent Co., Ltd., Shanghai, China. Spherical zinc powder (s-Zn) and flake zinc powder (f-Zn): industrial grade, particle size of 400 mesh, was purchased from Tianjin Renmai Metal Materials Co., Ltd., Tianjin, China. Silane coupling agent  was purchased from Kangjin New Material Technology Co., Ltd., Dongguan, China.Bisphenol A epoxy resin (E-51) with an epoxy value of 0.48~0.52, industrial grade, was purchased from Nantong Xingchen Synthetic Materials Co., Ltd., Nantong, China; Phenolic amine curing agent (T-31): industrial grade, amine value of 520 mg/KOH/g, was purchased from Guangzhou Yueqiang Chemical Co., Ltd., Guangzhou, China. Adipic acid (≥99.5%), an analytical reagent, was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. . The analytical reagent 1,4-butanediol (≥99.0%) was purchased from Xilong Scientific Co., Ltd., Shantou, China. P-toluenesulfonic acid (≥99.0%), an analytical reagent, was purchased from Tianjin Damao Chemical Reagent Factory, Tianjin, China. Triphenylphosphine  with a molecular weight of 1000 was prepared using the method of our laboratory, and epoxy-terminated polyester (E-CTPBA) was prepared by being epoxy-terminated at both ends. The resin matrix prepared by mixing 20 phr E-CTPBA and 100 phr E-51 was named CTPBA/EP. The detailed preparation method is set out in the literature .Mf-Zn and Ms-Zn were added to CTPBA/EP in concentrations of 0 phr, 5 phr, 10 phr, 15 phr, 20 phr and 30 phr , respectively. The high-speed mixer was fully stirred for 15 min. After 30 min of ultrasonic vibration, it was used as component A, and the amount of T-31 curing agent was 25 phr, which was used as component B. Mf-Zn-modified epoxy resin composites (Mf-Zn/CTPBA/EP) and Ms-Zn-powder-modified epoxy resin composites (Ms-Zn/CTPBA/EP) were prepared by mixing the two components, and all samples were vacuum defoamed and cured at 25 °C for 2 days. The preparation process is shown in The tensile properties and impact properties were tested according to GB/T 2567-2008. The tensile properties were tested at 25 °C at a speed of 10 mm/min.The impact properties were tested by non-notched impact. The plastic pendulum impact testing machine (PTM1000) was used for testing. The sample size was 120 mm × 15 mm × 10 mm.The tensile shear strength test refers to GB/T 7124-2008, and the SUS321 stainless steel sheet size was 100 mm × 25 mm × 1.6 mm. A single lap method was used. The length of the bonding surface was 12.5 ± 0.25 mm, the width was 25 ± 0.25 mm and the thickness of the adhesive layer was about 0.2 mm. The overflow adhesive was cleaned in time. After 2 days of curing, the universal tensile testing machine was used to test at 25 °C and 2 mm/min.The peel strength test refers to GB/T 2791-1995, and flexible material selected was stainless steel, with a size of 200 mm × 25 mm × 0.15 mm, 150 mm coating length and 100 mm/min speed stripping.−1, the resolution was 4 cm−1, and the scanning number was 32 times.A Fourier transform infrared spectrometer  was used to characterize the synthesized zinc powder before and after modification. The scanning range was 4000–400 cmThe contact angle of zinc powder before and after modification was measured by the contact angle measurement instrument SDC-80. The test solution was deionized water. The test method was the static drop method and the dosage was 3 μL each time.2.The thermal stability of the modified epoxy resin was tested by a thermos gravimetric analyzer . About 10 mg of the sample was weighed in the crucible, and the heating range was 25–700 °C, the heating rate was 10 °C/min and the gas atmosphere was NThe glass transition temperature of the sample was measured by a dynamic thermomechanical analyzer . The measurement mode was a single cantilever, the heating rate was 3 °C/min, the temperature range was 25–150 °C and the test frequency was 1 Hz.The scanning electron microscope  was used to test the morphology of Zn and the impact fracture of the cured substance. After gold-plating on the fracture surface, the accelerating voltage was 15 keV.An X-ray diffractometer  was used to characterize the zinc powder. Before the test, the zinc powder was dried in an oven at 120 °C for 1 h to remove moisture, and then the zinc powder was placed in a clean slide and flattened. The test was performed by compaction with another slide, with a voltage of 40 kV, a current of 30 mA, a scanning rate of 2°/min and a scanning angle of 2θ ranging from 5° to 90°. The light source was Cu-Kα ray, and the test target was a copper target.0 (accurate to 0.0001 g) before soaking was weighed by a precision electronic balance within 1 min. After soaking for a period of time, the sample was taken out, and the liquid on the surface of the sample was quickly wiped off with filter paper. The mass W1 after soaking was weighed (accurate to 0.0001 g), and the weighing was also completed within 1 min. The mass after soaking for t days was Wt, and the mass change rate after soaking for t days was calculated according to Equation (1):According to GB/T 1690-2010, the sample size was 25 mm × 50 mm × 2 mm. The sample was placed in an oven at 104 °C for 1 h, and after weighing, it was soaked in the medium  to maintain a constant temperature of 25 °C. The mass Wt was the mass change rate of the sample (%), W0 was the initial mass before soaking (g), Wt was the mass of the sample after soaking (g), and the result was the median value of the three sample values. According to GB/T 13353-92, the tensile shear strength samples of adhesives soaked in different media for 7 days were tested, and the strength change rate ∆δ  was calculated as Equation (2), where Δδ was the change rate of strength (%), δ0 and δ1 were the arithmetic mean values of the tensile shear strength in the blank test and after 7 days of immersion in the medium, respectively.C−1, which could be attributed to the presence of adsorbed water. There was a weak C–H absorption peak at 2925 cm−1, which might be due to the attachment of oil during the preparation process. The Si–O–Si antisymmetric stretching vibration characteristic absorption peaks of Ms-Zn and Mf-Zn appeared at a wave number of 1089 cm−1. In addition, the Zn–O characteristic absorption peaks appeared at 560 cm−1 and their peak positions migrated after modification. KH-550 had a Si–O bending vibration absorption peak at 460 cm−1, which also appeared in Ms-Zn and Mf-Zn. The above analysis and the following means of representation proved that KH-550 modified s-Zn and f-Zn successfully.The infrared spectra of s-Zn, Ms-Zn (KH-550-modified s-Zn), f-Zn and Mf-Zn (KH-550-modified f-Zn) were measured after grinding and tableting with KBr, as shown in The phases of f-Zn, Mf-Zn, s-Zn and Ms-Zn were analyzed by an X-ray diffractometer (XRD). The patterns are shown in Scanning electron microscopy (SEM) is a common characterization method for microscopic morphology analysis. In order to observe the morphological characteristics of zinc powder before and after modification and analyze its surface elements, SEM and an energy dispersive spectrometer (EDS) were used to observe. The SEM images of f-Zn and Mf-Zn are shown in Similarly, Contact angle was an important criterion to judge the quality of filler modification. The contact angle before and after modification of zinc powder is shown in The combination of filler and resin matrix can directly affect the performance of modified epoxy resin. Therefore, the effects of zinc powder treated with a coupling agent on the mechanical properties of carboxyl-terminated-polyester-modified epoxy resin was investigated. In this experiment, the addition of unmodified zinc powder (s-Zn and f-Zn) and KH-550-modified zinc powder (Ms-Zn and Mf-Zn) was 5 phr. The experimental results of the bonding strength and mechanical properties of the modified epoxy resin adhesive with the coupling agent treated and untreated fillers are shown in The adhesive properties and elongation at break of the epoxy resin adhesive (f-Zn/CTPBA/EP) with unmodified f-Zn decreased, and the tensile strength and impact strength increased to a certain extent. The mechanical properties and adhesive properties of epoxy resin adhesive (Mf-Zn/CTPBA/EP) with KH-550-modified f-Zn were improved compared with the pure CTPBA/EP matrix, and the values were higher than those of unmodified f-Zn. Relatively speaking, the bonding properties and mechanical properties of Mf-Zn/CTPBA/EP were better than those of Ms-Zn/CTPBA/EP. The above results showed that KH-550 had a certain promoting effect on the performance of the epoxy resin adhesive.The reason for the above phenomenon might be that the specific surface area of unmodified zinc powder was large, the surface was rich in hydroxyl groups, and the compatibility between inorganic particles and organic matter was poor, which was not easy to diffuse in the resin matrix, resulting in agglomeration, and a large number of defects appearing in the curing process, which reduced the comprehensive mechanical properties. After the zinc powder was modified by the silane coupling agent KH-550, the improvement of the comprehensive performance might be due to the particularity of the structure of the silane coupling agent, which could couple the zinc powder with the resin matrix, inhibit the tendency of the filler agglomeration, improve the interface bonding strength, improve the compatibility between the two, and effectively improve the comprehensive performance of the epoxy resin.As shown in As shown in The effect of the addition of Mf-Zn and Ms-Zn on the tensile strength and elongation at break of the CTPBA/EP epoxy resin is shown in 2, which was 91.11% higher than that without filler. This may be because when the amount of zinc powder added was less, it could be more evenly dispersed in the resin matrix, and the contact surface area with the epoxy resin was larger, which could improve the interfacial bonding between the nanoparticles and the resin matrix. At the same time, the filler could absorb a certain amount of energy when impacted, hinder the crack propagation of the resin and prevent it from developing into a destructive fracture, so as to achieve the purpose of toughening [The effect of Mf-Zn and Ms-Zn addition on the impact strength of CTPBA/EP is shown in ughening .The SEM images of the impact fracture surface of CTPBA/EP cured samples with different additions of Mf-Zn are shown in The energy dispersive spectrometer (EDS) was used to analyze and detect the elements of the surface exposed particles when the addition amount of Mf-Zn was 5 phr. As shown in 2 atmosphere. From 2 atmosphere. The addition of Mf-Zn significantly improved the thermal stability of the CTPBA/EP resin matrix. The initial temperature of thermal weight loss (IDT) and residual carbon rate increased with the increase in Mf-Zn content. The reason for this phenomenon might be that Mf-Zn itself was an inorganic particle, and its thermal stability was good. After adding the system, it formed a strong interaction with the CTPBA/EP resin matrix, so that the energy required for fracture was higher, resulting in improved thermal stability. The increase in the residual carbon rate may be due to the higher proportion of inorganic fillers; the lower the weight loss rate, the higher the residual carbon rate.Because the mechanical properties and bonding properties of Mf-Zn/CTPBA/EP were better than Ms-Zn/CTPBA/EP, Mf-Zn was selected as the research object. max corresponding to the maximum thermal weight loss rate moved in the high temperature direction. The data are shown in max of the Mf-Zn/CTPBA/EP was basically unchanged with the increase in the amount of filler added, all at about 380 °C.Tg) of the system increased. When the addition amount was 5 phr, the glass transition temperature increased rapidly from 70.3 to 75.6 °C, and the loss factor increased rapidly from 0.78 to 1.03. Tg decreased with the increase in the addition amount, but it was still greater than that of pure CTPBA/EP. This may be because the addition of the flake filler affected the movement of the polymer molecular chain and improved its damping performance. The flaky fillers were modified by KH-550 to improve the compatibility with the epoxy resin matrix. Due to the van der Waals force, it can form a molecular chain entanglement with the surface of the epoxy resin matrix, and play the role of a chemical cross-linking point in the system, which limited the fluidity of the chain segment, thus increasing the glass transition temperature [perature ,33.− caused an easy hydrolysis reaction with ester group destruction of cross-linked structure so that there was a small amount of curing off, so that the quality was reduced; the two reacted together so that the quality of the sample changed. The mass change rate of Mf-Zn/CTPBA/EP in various media was low, and the solvent resistance was good. This may be because Mf-Zn had a unique lamellar structure, which can be in close contact between the zinc powder and the matrix. The parallel overlap made the path of the corrosive substance in the medium to the interior more tortuous and longer, so that the mass change was smaller.A zinc-powder-modified epoxy resin was prepared by modifying a CTPBA/EP epoxy resin matrix with f-Zn and s-Zn, respectively. It had excellent bonding properties, mechanical properties and medium resistance. It had certain application value for adhesives that need to be used in harsh environments (such as seawater pipelines). Through the structure and morphology characterization of Mf-Zn and Ms-Zn and the bonding performance test, mechanical properties test, medium resistance analysis and impact section morphology characterization, the following conclusions were reached.2, which was 91.11% higher than that without filler. The addition of Mf-Zn could improve the thermal stability of CTPBA/EP. The glass transition temperature and loss factor tanδ increased. When the addition of Mf-Zn was 5 phr, Tg reached 75.6 °C, which was 5.2 °C higher than that without filler, and tanδ increased from 0.78 to 1.03. Compared with CTPBA/EP, Mf-Zn/CTPBA/EP had better medium resistance.The tensile strength, peel strength, tensile shear strength, impact strength and elongation at break of Mf-Zn and Ms-Zn modified by KH-550 were better than those of unmodified f-Zn and s-Zn. Relatively speaking, the effect of Mf-Zn-modified epoxy resin adhesive was better than that of Ms-Zn. The addition of Mf-Zn significantly increased the tensile shear strength and peel strength of the epoxy resin adhesive, while the addition of Ms-Zn decreased the tensile shear strength. Relatively speaking, the bonding performance of Mf-Zn-modified epoxy resin adhesive was significantly better than that of Ms-Zn. When the addition amount of Mf-Zn was 5 phr, the tensile shear strength of Mf-Zn reached a maximum value of 13.16 MPa, which was 15.95% higher than that without the addition, and the peel strength reached 0.124 kN/m, which was 55% higher than that without the addition. The addition of Mf-Zn and Ms-Zn significantly improved the tensile strength and impact strength of epoxy resin adhesive, and the tensile strength and impact strength increased first and then decreased with the increase in addition amounts and the elongation at break decreased with the increase in addition amounts. The mechanical properties of Mf-Zn-modified epoxy resin were significantly better than that of Ms-Zn. When the addition amount of Mf-Zn was 5 phr, the tensile strength of Mf-Zn reached a maximum of 43.09 MPa, which was 40.54% higher than that without filler, and the impact strength reached a maximum of 7.09 kJ/m


A CdS/CdTe solar cell device with thermally evaporated CuI/Au showed power conversion efficiency (PCE) of 6.92% with JSC, OCV, and FF of 21.98 mA/cm2, 0.64 V, and 0.49 under optimized fabrication conditions. Moreover, stability studies show that fabricated CdS/CdTe thin-film solar cells with CuI hole-transporters have better stability than CdS/CdTe thin-film solar cells with Cu/Au back contacts. The significant increase in FF and, hence, PCE, and the stability of CdS/CdTe solar cells with CuI, reveals that Cu diffusion could be avoided by replacing Cu with CuI, which provides good band alignment with CdTe, as confirmed by XPS. Such an electronic band structure alignment allows smooth hole transport from CdTe to CuI, which acts as an electron reflector. Hence, CuI is a promising alternative stable hole-transporter for CdS/CdTe thin-film solar cells that increases the PCE and stability.This study focuses on fabricating efficient CdS/CdTe thin-film solar cells with thermally evaporated cuprous iodide (CuI) as hole-transporting material (HTM) by replacing Cu back contact in conventional CdS/CdTe solar cells to avoid Cu diffusion. In this study, a simple thermal evaporation method was used for the CuI deposition. The current-voltage characteristic of devices with CuI films of thickness 5 nm to 30 nm was examined under illuminations of 100 mW/cm Furtherve to Cu ,7,8,9. Tve to Cu ,11,12. HCopper iodide has suitable properties that contribute to its application as an HTM in solar cells, such as a wide band gap (Eg) of 3.1 eV and stable p-type conductivity at room temperature. Depending on the thermal stability, the crystal structure of CuI is classified into α, β, and γ-structural phases. The α-phase is a cubic structure with a high temperature of 392 °C, the hexagonal β-phase is an ionic conductor with a temperature range of 350–392 °C, and the γ-phase is a cubic structure with a low temperature, below 350 °C . Mainly,−2 Ωcm [CuI films are prepared using various techniques, such as solution process deposition /n-CdS/p-CdTe/CuI/Au. FTO-coated substrates  were cleaned as reported elsewhere [2 environment at 375 °C for 30 min. Thereafter, ~5 μm CdTe absorber layer was deposited on CBD-CdS by close-spaced sublimation (CSS) system , with source and substrate temperatures of 640 °C and 580 °C, respectively, under various chamber pressures of 5.0 Torr and 7.9 torr in an Argon medium to investigate the effect of CSS conditions for CdTe layer. In order to investigate the effect of a CuI hole transport layer on CdTe solar cells, CuI films with thicknesses ranging from 5 nm to 30 nm were deposited on the CdTe film by thermal evaporation . Following the CuI film deposition, an ~80 nm thick Au layer was thermally evaporated. CBD-CdS/CSS-CdTe/Cu/Au devices were also fabricated as a comparison to demonstrate beneficial effects induced by a CuI hole-transporter. All the fabricated devices were later annealed at 200 °C for 10 min in N2 environment.The substrates or films were kept in an open environment except during fabrication. The CdS/CdTe thin-film solar cells were fabricated with structure of glass/SnOlsewhere . Window lsewhere ,45,46,472 with an Air Mass of 1.5 filter.The electrical properties of CuI were measured using the four-point probe (FPP) technique . The optical and structural properties of each layer were characterized by using UV-Vis , XRD  spectroscopy, and Atomic Force Microscopy . XPS analysis was performed using the Thermo Scientific ESCALAB Xi instrument  with Al Kα as the X-ray source. Finally, their photovoltaic performance and device stability were analyzed using a solar simulator  under the illumination of 100 mW/cmThe chemical-bath-deposited CdS surface has low roughness of 33.60 nm (RMS of 8.42 nm). This was further evidence for higher transmission of the CdS layer due to the lower light scattering. As the roughness of the CdS window layer, as shown in The electrical resistivity of the thermally evaporated CuI film was measured by using the four-point probe measurement technique (FPP) , which iWhen the probe distance d is much larger than the film thickness, The sheet resistance and resistivity of fabricated CuI were measured as 19.856 kΩ/sq and 0.20 Ωcm, respectively , at room ~3.0 eV . The ban ~3.0 eV ,51,52 an ~3.0 eV ,54.The band alignment of the CdTe/CuI interface is important to the hole transport and formation of low electronic resistance, as it enhances the hole transport and reduces carrier recombination. Based on the XPS measurements, the valance band offset value of The current-voltage characteristic curves of CBD-CdS/CSS-CdTe solar cells with different thicknesses of the CuI hole transport layer are shown in S), which increases with CuI thickness due to the high resistivity in the thicker CuI layer preventing the carrier transport. The devices with 10nm of CuI thickness deliver the best performance due to the lowest RS and largely improved JSC, RSH, and FF, compared to those devices with lower and higher thicknesses. This is mainly due to the higher hole concentration in CdTe. Higher hole concentration can boost a higher build-in potential (Vbi) and lead to less recombination in the depletion region and at the front interface, thereby resulting in larger FF, which can be further confirmed through an improvement in the shunt resistance (RSH). Roussillon et al. also suggested that the back contact barrier on the properties of CdTe solar cells depend on the space charge region in the main CBD-CdS/CSS-CdTe junction and the space charge region in the back contact may overlap, depending on the barrier height of the back contact [2 for 5 nm to 994 Ωcm2 for 10 nm thickness of CuI. When the thickness of CuI was further increased to 30 nm, the RSH decreases to 186 Ωcm2. The JSC also shows a similar trend as the FF and the shunt resistance with the CuI hole-transport layer thickness. A similar trend was observed in a reported study [The short circuit current density is influenced by the series resistance  [−9 A/cm2 and 1.5 × 10−7 A/cm2, respectively. This means the dark saturation current density in the device with Cu/Au back contact is nearly two orders of magnitude lower than that of the device with CuI/Au back contact. The CuXTe structure is formed at the back of the CdTe absorber by the diffusion of copper, as mentioned earlier. Most of the Cu diffused into the bulk finds its way to the CdS with time, lowering its space charge density and causing it to become photoconductive. This effect of Cu in the CdTe is more problematic, adding recombination centers (lowering the efficiency) and increasing the space charge density, which might either raise or lower efficiency, depending on CdTe thickness [SC of the device with a CuI hole-transporter was significantly higher than the device with Cu/Au back contacts, which is due to the hole transport property of the thermally evaporated CuI and band alignment between the CdTe and CuI. The fill factor of the devices with a CuI/Au was significantly higher than that of the Cu/Au devices, which was induced by the smaller RS and larger RSH value of the device with a CuI/Au back contact than Cu/Au back contact.The measured photovoltaic performances of the fabricated devices instantly after fabrication and after one month for devices with 10 nm Cu/Au bi-metal and CuI/Au back contacts are shown in  studies ,67,68. Vtion (6)  and can hickness . This is2 with AM 1.5 filter and in dark. The influence of different growth conditions of CSS-CdTe on the photovoltaic performance of the device was also analyzed with CSS-CdTe growth conditions of 5.0 torr and 7.9 torr, which were fabricated with a 10 nm CuI hole-transporter. 0) was SC, OCV, and FF of 21.98 mA/cm2, 0.638 V and 0.49, respectively. OCV and FF in a generic FTO/CBD-CdS/CSS-CdTe/back-contact thin-film solar cell device is a key parameter in the recombination analysis. In particular, OCV is sensitively influenced by the interface recombination at the buffer/absorber, front interface, and the absorber/back-contact interface [This also shows the roll-over phenomenon disappeared in CdTe solar cells fabricated under 7.9 torr. Further, a later crossover of the light and dark J–V curves for the cell fabricated under 7.9 torr demonstrates that the energy barrier at the back contact was reduced, and the estimated reverse saturation diode current .V = 0) recombination coefficients at the buffer/absorber interface, in the depletion region (Wd), in the quasi-neutral region, and at the absorber/back-contact interface, respectively [OCV depends on the carrier recombination of the solar cells influenced by the fabrication conditions of each layer. Fabrication conditions affected the grain size (The highest ectively . This meain size , surfaceain size . Only leain size . This shThermally evaporated CuI was successfully employed as a hole-transporter for CBD-CdS/CSS-CdTe solar cells. A significant improvement in fill-factor and, hence, efficiency was achieved by replacing Cu as back contact with CuI. Structural and opto-electrical properties of thermally evaporated CuI reveal that the deposited film has γ-phase CuI, which has good electrical conductivity and well-matched energy band alignment with CSS-CdTe. Further, XPS studies of the CdTe/CuI interface confirmed that a small valence band offset at the CdTe/CuI interface improves the photo-generated hole transport from CdTe to CuI without any barrier, and the large conduction band offset between CdTe and CuI reduces the electron transport from back contact to CdTe; thus, the insertion of CuI as a hole-transporter can reduce the electron recombination rate at the back contact. The results clearly showed that CuI is a promising hole-transporter instead of Cu as back contact for CdS/CdTe thin-film solar cells, as that will prevent Cu diffusion, increase stability, and also increase the PCE considerably.


Ms) was 0.79 × 10−4 emu/g, 1.28 × 10−4 emu/g, and 1.5 × 10−4 emu/g for (110), (100) and (111) orientation implanted-MgO and follows the relation Ms(111) > Ms(100) > Ms(110), indicative of crystalline orientation-dependent ferromagnetism in N-implanted MgO. The samples were characterized by X-ray photoelectron spectroscopy (XPS), high resolution X-ray diffraction (HRXRD), reciprocal space mapping (RSM), and photoluminescence (PL). The results indicated that the amount of N-substitute-O and N-interstitial defects in these three N-implanted MgO samples showed the same changing tendency as compared with Ms data. Thus, we conclude that the N-substitute-O and N-interstitial defects may play a crucial role in controlling the N+-implanted-induced ferromagnetism.Samples of (110), (100), and (111) MgO single crystals were implanted with 70 keV N ions at room temperature. All as-implanted samples showed room temperature hysteresis in magnetization loops. The observed saturation magnetization ( Due to the prospect of fabricating faster and more energy-efficient circuits than standard semiconductor devices, spintronics have attracted continuous attention in the past several decades. Some research studies have reported novel achievements that can provide new forms of functionality, such as spin field-effect transistors, giant magnetoresistive sensors, storage media, etc. ,2,3. The2 [2 nanoparticles [To overcome these limitations, the RTFM was improved by doping nonmagnetic elements in traditional semiconductors . Along t2 , Ag-dope2 , and Li-articles , etc. Alarticles , doping articles , and annarticles , are useMagnesium oxide (MgO) is considered to be a promising material in the field of spintronic devices due to its wide band gap, optical behavior, and multilevel switching characteristics . TherefoIon implantation is a promising tool in the field of defect engineering due to its controllable, reproducible, efficient, and non-equilibrium characteristics. The implanted ions may change the crystal structural properties and electrical properties due to the energetic collision cascades, and endow unexpected properties to the materials. In the case of MgO, numerous experiments on ion implantation have been done. G. Baubekov et al.  deeply iIn the current paper, we implanted N ions into MgO single crystals with (100), (110), and (111) orientations and successfully observed orientation-dependent ferromagnetism. To clarify the orientation-dependent ferromagnetism, the microstructures of the pristine and as-implanted samples were characterized in detail. Our results indicate that defect type was crucial in introducing ferromagnetism in MgO. The discovery may provide a valuable reference for controlling ferromagnetism in oxide semiconductors.17 cm−2 at room temperature. Samples were placed on a wafer tray during the process of ion implantation to avoid contact with metal support. The implantater was performed at an angle of 7° off normal direction to decrease the channeling impact. The base pressure of the chamber was maintained at 2 × 10−4 Pa. Qualitative analysis of the elements experiment was carried out by X-ray photoelectron spectroscopy  with monochromatic Al Kα X-ray radiation. The magnetic measurements data were obtained through a superconducting quantum interference device  at room temperature. The applied magnetic field was parallel to the sample surface. Both before and during the process of magnetic measurement, the operation was conducted carefully to avoid magnetic impurities contamination. The microstructures of pristine and as-implanted samples were characterized in Beijing Synchrotron Radiation Facility (BSRF) with the X-ray wavelength of 0.15493 nm. The high-resolution X-ray diffraction (HRXRD) and reciprocal space mapping (RSM) were measured with a scanning step width of 0.02°. The photoluminescence (PL) emission spectra were measured by spectrofluorometer  using the 370 nm laser diode (LD) as an excitation source.MgO single crystals with (100), (110), and (111) orientations were purchased from Hefei Kejing Materials Technology Co., Ltd. . The samples were prepared using the arc melting method. The samples were cut into the size of 10 mm × 5 mm × 0.5 mm. An ion implantation experiment was conducted at the Institute of Semiconductors, CAS, Beijing. N ions with the energy of 70 keV were implanted into MgO samples with the influent of 2 × 10Ms) shows apparent crystallographic orientation dependence. The magnitude of saturation magnetization of as-implanted samples with different orientations is clearly presented in Ms) in as-implanted samples with (111) orientation is the largest, at about 1.5 × 10−4 emu/g, and the smallest saturation magnetization emerged in the sample with (110) orientation, at about 0.79 × 10−4 emu/g, which is twice as small as the largest one. Obviously, such a large difference cannot be a measurement error. Normally, several factors could influence ferromagnetism in doped systems, including crystallization, doped environment, and implantation dose, etc. In our experiment, the above variables were unified. Theoretical and experimental research studies propose that the magnitude of saturation magnetization is determined by the defect concentration [Mg0, VMg−, VMg2−) and O vacancy . A review article [Mg0 and VMg− are responsible for the magnetic moment, while the VMg2−, VO0, VO+ and VO2+ have no contribution to the magnetic moment. Furthermore, a previous study demonstrated that the amount of displaced atoms and damage in Ar+-implanted MgO depends on the crystallographic orientation [+-implanted MgO. The amounts of displaced atoms increased from (111) through (100) to the (110) orientation, which is consistent with the rising tendency of Ms in different orientations. Such a difference in the number of displaced atoms should result in the concentration difference of specific defect types. Based on the above results, we speculate that the number of certain defect types may be a factor in explaining the observed orientation-dependent ferromagnetism.The magnetic field-dependent magnetization (M-H) data were measured at room temperature to investigate these samples’ magnetic properties. ntration , but the article  summarizentation . The abo+-implanted samples. All the N 1s peaks of the N+-implanted samples can be fitted into two symmetric peaks, with Na located at 398.9 eV~399 eV and Nb located at about 398.8~399.1 eV. The Na can be obviously assigned to α-N2 [b peak can be ascribed to N-related defects. Noticeably, the relative peak area of Nb increases from (110) through (100) to the (111) orientation, and the value is 41%, 56.8%, and 62.1%, respectively. The relative peak area of Nb is positively associated with the magnitude of the magnetic moment. Hence, we conclude that the N-related defects play a key role in inducing the ferromagnetism in N+-implanted MgO. The most possible situation is that N ions substitute O ions (NO) or N ions locate at the interstitial site (Nint) even though the existence of vacancy-Mg and vacancy-O and Interstitial-O are prior to NO and Nint in terms of formation energy [The XPS was carried out to clarify the difference in defect composition in these pristine and implanted samples. In spite of the magnetic measurement process being conducted carefully, the XPS was still used to exclude possible extraneous magnetic impurities, such as Fe, Co, Ni, Mn, and Cr. The XPS full spectrum is shown in  to α-N2 . Referri to α-N2 , the Nb n energy . The unpy and qz are the horizontal and vertical component of the scattering vector, respectively. According to diffuse scattering theory, defects can give rise to lattice distortions, and the long-range part of this distortion leads to the scattering vector deviating from the reciprocal lattice points [+-implantation, the shape of scattering diffuse distribution has no significant change, which suggests that the intrinsic defects are still dominate. On the other hand, for +-implanted (110), (100), and (111) orientation samples along the qy-axis are displayed in To further investigate the defect types in as-implanted MgO, we measured RSMs of 200, 220, and 111 reciprocal lattice points of the pristine and as-implanted MgO samples with (100), (110), and (111) orientation, respectively. The results are shown in e points . Simultae points . Figure +-implanted samples. As shown in the inset, all samples present a single crystal structure, since no additional second-phase diffraction peaks were detected. Additionally, the diffraction intensity of all N+-implanted samples decreases compared to the pristine samples, which is indicative of the lattice disorder resulting from the implantation-induced defects. Remarkably, all the diffraction peaks move to a lower angle position in as-implanted samples compared with the pristine MgO. The value of peak shift increases from (110) to (100) to the (111) orientation by 0.078°, 0.237°, and 0.278°, respectively. The peak left-shift is related to the lattice expansion. In MgO, the lattice expansion likely originates from Nint or NO [int and NO defects. This result is consistent with the RSM results.In order to get more defect-related information, high-resolution X-ray diffraction was used to characterize the microstructure. nt or NO . As is k+ (oxygen vacancy with one electron) center, which is originated from the 2T1u → 2A1g transition [3B1u → 1Ag transitions of the F22+ (combinations of neighboring F centers with two electrons) center [The PL spectra were measured at room temperature. As shown in ansition . The emi) center . The emi) center . Howeverint and NO defects are orientation-dependent in N+-implanted MgO single crystals. The concentration of N-related defects decreases from (111) through (100) to (110) N+-implanted MgO. This is probably due to the distinctive properties in the different crystallographic planes. The charge and atomic density distributions existing on the (110), (110), and (111) planes are dissimilar. Usov et al. [+-implanted MgO, the N-related defects are more likely generated in (111) and (100) oriented samples than in the (110) oriented sample due to the weaker damage resistance and higher defect migration efficiency.Based on the above experimental results and literature description, we can draw the conclusion that the concentration of Nv et al.  reportedv et al. . Mao et v et al.  investigThe observed value of saturation magnetization is subject to the same changing tendency of N-related concentration. This result demonstrates that the N-interstitial and N-substitute-O defects introduce ferromagnetism and enhance the magnetic moment. Both of these two defect types can contribute unpaired electrons from N 2p orbitals, and produce the localized spin magnetic moment. Moreover, according to the Heisenberg exchange model, the coupling between the unpaired electrons from the N-related defects and intrinsic vacancies can result in local magnetic ordering through exchange correlation. This should be the source of ferromagnetism.+-implanted MgO single crystals with (100), (110), and (111) orientation. The saturation magnetization presents an orientation-dependent characteristic. The RSM, XPS, HRHRD, and PL results demonstrated that the concentration of N-substitute-O and N-interstitial defects has a similar orientation-dependent characteristic with the magnetic properties. The orientation-dependent concentration of N-related defects may be due to the distinctive properties in the different crystallographic planes. Consequently, the localized magnetic moments induced by N-related defects were also discussed.In summary, apparent RTFM was observed in N


Few studies have implied the incidence of diaphragmatic hernia (DH) after spontaneous pneumothorax (SP) with unknown mechanisms. The current study aimed to identify whether there is an association between the DH and SP.We selected 46,897 patients with SP (SP cohort) and 46,897 without SP (non-SP matched cohort) from the National Health Insurance Database. Patients were frequency matched according to age, sex, and index year. The incidence of DH and its association with SP were assessed after stratifying different characteristics and comorbidities. Statistical analysis including chi-square test, t-test, cox proportional hazard model, and Kaplan–Meier method were used.The results suggested there were significant associations between SP and DH, especially in the subgroup of patients with older age (aged 40–64 years: 2.61-fold in adjusted hazard ratio (aHR), 95% confidence interval (CI): 1.27–5.36; aged > 65 years: 1.97-fold in aHR, 95% CI 1.43–2.71), male sex , hypertension , diabetes mellitus , and smoking-related disease . The SP cohort has significantly correlated with DH within 5-year follow-up .The SP cohort had a higher incidence of DH than the non-SP matched cohort. A prospective study of indications based on the findings of the current research should be performed. In a global estimate, the incidence of spontaneous pneumothorax (SP) is 7.4–18 cases per 100,000 population each year in males, and 1.2–6 cases per 100,000 population each year in females . The tobIn addition to iatrogenic diaphragmatic hernia (DH) , 6, congAccumulating data have shown that the SP may be associated with the development of DH –10. The The Taiwan government established the National Health Insurance Research Database (NHIRD), which has a high health coverage rate and included the health claim data of nearly 99% of the Taiwan population from the Taiwan NHI. This study conducted analyses using data from the population-based hospitalization database, which included more than 20 million participants with available data from 1996 to 2013. The identification number of each patient was encrypted to ensure privacy. The diagnoses in the Taiwan NHI are coded according to the International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM). The current study was approved by the research ethics committee of China Medical University and Hospital in Taiwan (CMUH-104-REC2-115-(CR6)) and performed in accordance with the Declaration of Helsinki.To validate the association between SP and DH, we conducted a retrospective cohort study. The participants were classified into the SP and non-SP matched cohorts. Patients with SP who were aged older than 20 years had at least one hospitalization due to SP (ICD-9-CM: 512.0 and 512.8) from 2000 to 2012. The index date was set as the first SP diagnosis date. Subsequently, patients diagnosed with DH before the indexed date or aged < 18  were excluded from this study. The surveying purpose of this study was DH (ICD-9-CM codes 551.3 (diaphragmatic hernia with gangrene), 552.3 (diaphragmatic hernia with obstruction), and 553.3(diaphragmatic hernia)). Patients with at least one hospitalization diagnosis were included. The follow-up period was from the index date to the first diagnosis of DH, withdrawal from the NHIRD, or until December 31, 2013. For each patient with SP, we assigned one who was never diagnosed with SP via propensity score matching. The matching variables were age; sex; index year; and comorbidities including hypertension (ICD-9-CM codes 401–405) , diabeteThe categorical variables were sex, age , and comorbidities, and they were presented as numbers and percentages. Age was expressed as mean and standard deviation, and the difference between the two cohorts was assessed using the chi-square test and the t-test.p value of < 0.05 was considered statistically significant.The association between SP and the risk of DH was evaluated using the cox proportional hazard model. The results were presented as hazard ratios (HR), adjusted hazard ratios (aHR), and 95% confidence intervals (CIs). The Kaplan–Meier method was used to identify the cumulative incidence curves of DH between the SP and non-SP matched cohorts. The difference between two curves was assessed using the log-rank test. All statistical analyses were performed using SAS statistical software version 9.4 . The results were plotted using R software. A two-sided p value < 0.05).Each cohort comprised 46,897 eligible participants Table . Among tTable After stratification with variables Table , patientWhen stratified by follow-up years, the SP cohort who were followed up for less than fivers years from the index date showed a significantly higher risk of DH  (Table p = 0.003).The SP cohort had a significantly higher cumulative incidence of DH than the non-SP group in Fig. The results in this study found the SP cohort had a significantly higher cumulative incidence of DH than the non-SP group in the 10-year follow-up. The risk factors of DH include SP, older age, and smoking related disease. After stratification with various subgroup, the SP cohort with older age (aged > 40 years), male sex, hypertension, diabetes mellitus, and smoking-related disease had signification associations with the DH.There may be controversies regarding whether the DH was congenital or caused by trauma. To prevent bias, the patients with DH who had a previous history of trauma within the last 5 years were not included to prevent including those suspected with congenital DH.This study showed that the SP cohort had a higher incidence of DH than the non-SP matched cohort, one proposed mechanism might be attributed to possible collagen fiber defect which resulted from systemic collagenase effect , 19. AltThere were significant associations between SP and DH in the subgroup of patients with older age (aged > 40 years), male sex, hypertension, diabetes mellitus, and smoking-related disease. Table . A previOur data suggested the SP was significantly correlated with DH within 5-year follow-up, but not in follow-up longer than 5 years Table . The detTo the best of our knowledge, this is the first large-scale and long-term follow-up study showing the association between SP and DH. At the end of the 10-year follow-up, the SP cohort had a significantly higher cumulative incidence of DH than the non-SP matched cohort , male sex, hypertension, diabetes mellitus, and smoking-related disease, had a higher incidence of DH than the non-SP matched cohort. In addition, the SP was significantly correlated with DH within 5-year follow-up. A prospective study of indications based on the findings of the current research should be performed.


The mean age of the studied population was 61.61 ± 12.95 years, BMI 25.72 ± 3.82 Kg/m2, FEV1/FVC 68.37 ± 12.00%, and FEV1 80.07 ± 23.63% predicted. According to microarray analysis, three miRNAs of the most upregulated were chosen: miR-320c, miR-200c-3p, and miR-449c-5p. These miRNAs were validated by qPCR and were shown to be differently expressed in COPD patients. ROC analysis showed that these three miRNAs together had an area under the curve of 0.89 in differentiating COPD from controls. Moreover, in silico analysis of candidate miRNAs by DIANA-miRPath showed potential involvement in the EGFR and Hippo pathways. These results suggest a specific 3-miRNA signature that could be potentially used as a biomarker to distinguish COPD patients from smokers and non-smoker subjects.Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease commonly induced by cigarette smoke. The expression of miRNAs can be altered in patients with COPD and could be used as a biomarker. We aimed to identify a panel of miRNAs in bronchoalveolar lavage (BAL) to differentiate COPD patients from smokers and non-smokers with normal lung function. Accordingly, forty-five subjects classified as COPD, smokers, and non-smokers ( Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death worldwide and expected to increase in the near future . Since mWe hypothesized, therefore, that changes in the level of certain miRNAs could be used as specific biomarkers in patients with COPD. Accordingly, in this study, we aimed to develop a panel of miRNAs in BAL that would allow us to detect and differentiate patients with COPD from the smokers and non-smokers. To this purpose, we performed a single-center, cross-sectional case-control in 45 subjects that were divided into COPD, smokers, and non-smokers with normal lung function to study the miRNA profile in BAL. Furthermore, we carried out an in silico analysis to study which pathological processes might be regulated by the candidate miRNAs that were found differentially expressed in the COPD group.We present a single-center, cross-sectional case-control study that recruited patients at the Hospital Universitari Son Espases  who had clinical indications for bronchoscopy: mostly hemoptysis, lung nodule, or lung mass that later resulted negative for infection or lung cancer. The patients were then classified into groups based on the results of bronchoscopy, chest computed tomography (CT) (+/− fine needle aspiration), and spirometry. The project was approved by the Ethics Committee of the Balearic Islands in Spain.Participants were selected from individuals referred to the pneumology outpatient clinic and bronchoscopy areas. To group the individuals according to their smoking status, we used the term “pack-year”, defined as the number of packs smoked per day multiplied by the number of years the person has smoked. The non-smoker group included patients with normal lung function and zero pack-years. The smokers with normal lung function and COPD patients with a cumulative smoking exposure of at least 10 pack-years were selected for the study. Participants were grouped by gender and age. At the time of recruitment, all COPD patients were stable, which is defined by the absence of exacerbations and stability in treatment for the last three months. COPD diagnosis and its severity were defined using post-bronchodilator spirometry indicators according to the Global Initiative in Obstructive Lung Diseases (GOLD) recommendations . The exc8 copies of UniSp6 RNA Spike-in control  were added to BAL before RNA isolation. UniSp6 RNA Spike-in control and the circulating miRNA miR-16-5p were used for monitoring both RNA isolation and cDNA synthesis procedures by quantitative PCR (qPCR) [Following the manufacturer’s recommendations to use the same starting volume of biofluid to ensure consistent RNAinput, total RNA, including the miRNAs fraction, were isolated from 200 μL of BAL with the miRNeasy advanced serum/plasma kit  according to the manufacturer’s protocol. A total of 10n = 5) and smoker (n = 5). Both groups were matched by sex. Accurate miRNA isolation was previously confirmed by qPCR for UniSp6 RNA Spike-in control and for the endogenous miR-16-5p. Cq values were consistent amongst all 10 samples. We used 8 μL of total RNA for direct labelling with the FlashTagTM Biotin HSR Labeling Kit, and the labelled samples were hybridized at 48 °C and 60 rpm for 42 h to the GeneChip® miRNA 4.0 Array . Array normalization was executed with the Transcriptome Analysis Software 4.0  following the robust multichip analysis (RMA) and the detected above background (DABG) algorithms. The eBayes method was used to identify the differentially expressed miRNAs amongst groups and were considered biologically significant if they displayed a fold change value  and p-value < 0.05. DIANA-miRPath v3.0 was used for KEGG pathway enrichment based on predicted miRNA targets provided by the DIANA-microT-CDS algorithm [A discovery set of samples were selected from patients for microarray analysis. Two groups were considered: COPD (lgorithm . We studn = 15 each). We used the miRCURY LNA Universal RT microRNA PCR System  for the detection of miRNAs by quantitative real-time PCR (qPCR) using SYBR green. According to the instructions of the reverse transcription reagent, 2 µL of total RNA was reverse transcribed into cDNA with miRCURY LNA RT Kit  and incubated for 60 min at 42 °C, 5 min at 95 °C to heat inactivate the reverse transcriptase, and stored at 4 °C. cDNA was diluted 20× with nuclease free water. Subsequent real-time PCR reactions were performed in duplicate with the miRCURY LNA SYBR Green PCR Kit  with LNA-specific primers using a CFX96 system . The thermocycling conditions were as follows: 95 °C for 2 min, followed by 44 cycles of denaturation at 95 °C for 10 s, and annealing/extension at 56 °C for 1 min. Product specificity was confirmed in initial experiments by melting curve analysis, and PCR efficiency was calculated for each LNA specific primer. The expression of the housekeeping gene (miR-16-5p) was used as an internal control to normalize the expression of each target gene. Relative quantification with the 2−∆∆Cq method [Candidate miRNAs were validated in the complete cohort of 45 patients  and percentage (%), as appropriate. Comparison between experimental groups was performed using analysis of variance (One-way ANOVA) followed by Bonferroni post hoc to compare different groups. A p-value of <0.05 was considered to represent a statistically significant difference. All statistical analysis was carried out using GraphPad InStat version 9.00 .All the data were presented as mean ± standard deviation (SD) or number . Twenty-one of them (46.67%) were men, and twenty-four (53.33%) were women. The mean age of the studied population was 61.61 ± 12.95 years, BMI 25.72 ± 3.82 Kg/m Disease , patientA discovery set of 10 samples for the groups of COPD and smoker was pre-selected for microarray analysis. Principal component analysis of the normalized signal values showed that COPD and smoker samples appeared to be consistent among the individuals in each group, thus distributing the samples into two differentiated clusters A. Signifp = 0.03) and non-smokers (0.0025) (p = 0.0218) and non-smokers (p = 0.0064) (p = 0.002) and non-smokers (p = 0.0112)  A, miR-20 0.0064) B, and mi 0.0112) C. In sump < 0.05) discriminated COPD patients from the smoker group with an AUC of 0.87 and 0.74, respectively. On the other hand, miR-320c could not discriminate significantly (AUC of 0.69)  and by the ROC curve  indicated that the use of the three miRNAs together as biomarkers may be suitable for COPD  A. A multfor COPD B.p-value = 0.0005). Contrary to our results, miR-200c was found to be downregulated in one case . Moreover, miR-320 family was overexpressed  in COPD patients . Therefore, this analysis allowed us to qualify two out of our three candidate miRNAs in other independent cohorts.Finally, we carried out an in silico analysis to study the potential targets and pathways that are regulated by the three newly identified miRNAs. Firstly, the validated targets were identified in BAL from COPD, smokers, and non-smokers . The functional enrichment analysis reported that the three miRNAs regulate different pathways that are involved in COPD processes, such as adherents junction, ErbB signaling pathway, Notch signaling pathway, and Wnt signaling pathway (miR-449c-5p); PI3K-Akt signaling pathway, p53 signaling pathway, tight junction, Hippo signaling pathway, ErbB signaling pathway, ubiquitin mediated proteolysis, TGF-beta signaling pathway, and Wnt signaling pathway (miR-200c-3p); and Hippo signaling pathway, cellular senescence, and mTOR signaling pathway (miR-320c) . This anCOPD is characterized by an abnormal inflammatory response of the lungs caused mainly by tobacco. The decrease in expiratory airflow and the emphysema progresses along with severity even after the patient has stopped smoking . The disThere is some evidence that in COPD, lung and blood cells express different amounts of miRNAs compared with healthy controls . In factp-value and selected three candidate miRNAs, namely miR-200c-3p, miR-449c-5p, and miR-320c, for qPCR analysis including the complete cohort of 45 patients and confirmed that they were significantly upregulated in the COPD cohort compared to smokers and non-smokers. It must be mentioned that among all significantly differently expressed miRNAs detected by microarray analysis, the top ten candidates were all upregulated in COPD (including our three candidates). However, since miRNAs suppress gene expression, those that are significantly downregulated might also be interesting to study, as this results finally in upregulation of the target genes.Here, we studied the miRNA profile in COPD, smokers, and non-smoker individuals to identify alterations that might allow to differentiate the COPD group from the rest. In an initial microarray analysis that included ten patients, we found that 51 miRNAs where downregulated, whereas 93 miRNAs appeared upregulated in the cohort of well-characterized COPD patients compared to smokers. We then decided to focus on those that exhibit a greater Despite tobacco being the most common cause of COPD, not all the smoker population will eventually develop the disease. Therefore, it might be possible that smoking is upregulating specific miRNAs in some of the smokers, thus conferring an additional risk for COPD. In fact, several authors have shown that tobacco smoke causes changes in gene expression of respiratory epithelium, which is linked to the development of respiratory diseases, such as COPD ,19,21. CApart from miR-200c-3p and miR-449c-5p, we also observed a significant increase in miR-320c in BAL from COPD patients compared to smokers and non-smokers. Similar to us, another study also found upregulated miR-320c in plasma from COPD patients and also in asthma-COPD overlap disease . In addiIn addition, let-7c has been reported to decrease with cigarette smoke exposure in a rat model of COPD , to be dExtracellular vesicles (EVs), such as exosomes, are small vesicles that can be secreted into the BAL and transport a variety of components, including miRNAs, and have been recently described to play a role in COPD . MoreoveThe miRNAs that we observed differently expressed in COPD have also been found dysregulated in other inflammatory chronic lung diseases. For instance, whereas we observed upregulation of miR-320c in BAL from COPD subjects, downregulation in plasma from bronchial asthma patients has been reported by others ,32. In lDeregulation of miRNAs translates into altered expression of molecules and subsequent alteration in the pathways involved. Differences in the expression of miRNAs involved in the remodeling of the extracellular matrix have been reported in several chronic lung diseases , and in BAL is a heterogeneous sample where several cell types can be found, including immune cells and epithelial cells; moreover, extracellular vesicles coming from any cell type present in the lung can potentially end up in the BAL as well . TherefoThe size of our cohort was limited since this was a pilot study that aimed to give some preliminary insights in the miRNA profiling of BAL from COPD patients. To increase the reliability of our study, we conducted additional bioinformatics analysis in other similar studies, which confirmed upregulation of two out of our three candidate miRNAs (miR-449 and miR-320) in COPD. Nevertheless, the use of a larger sample size, especially in the discovery phase, will provide more result accuracy.p < 0.001). In this regard, the study from Bersimbaev et al. reported a ROC curve of COPD patients for plasma levels of miR-320c with an AUC of 0.855. Although the values are closed to ours, we could not find a significance for miR-320c upon ROC analysis, which might be influenced by the limited size of our cohort and by the fact that Bersimbaev et al. used plasma samples instead of BAL. [The limited size did not allow us to correlate the expression of our miRNAs with disease severity; however, we could evaluate the diagnostic potential of our three miRNA candidates by plotting ROC curves and found that miR-200c-3p and miR-449c-5p were both capable of discriminating COPD from smokers. Moreover, greater sensibility was reported when analyzing the three miRNAs, namely miR-200c-3p, miR-449c-5p, and miR-320c, as a group (AUC = 0.89,  of BAL. .This study has analyzed the miRNA profile in BAL from COPD, smokers, and non-smoker individuals; however, miRNAs can also be extracted from liquid biopsies, such as blood collection. In fact, dysregulated miRNAs have been identified in lung tissue and other body fluids in COPD, including plasma, serum, sputum, bronchial lavage, saliva, and pleural fluid, compared to normal lung ,19,20. ESome of our findings slightly differ from previous studies; however, it needs to be considered that others have mostly used lung tissue to study the miRNA expression profile in COPD, whereas we used BAL, which contains material from a heterogeneous cell population. On the other hand, identifying new miRNA profiles in the early stages of disease that are specific for COPD in comparison to smokers could be used as a potential tool to predict which smokers could potentially develop COPD, thus facilitating the early treatment and monitoring of disease.This pilot study included a cohort of subjects classified in COPD, smokers, and non-smokers that were subjected to miRNA profiling in BAL samples. Despite the limited size, our results suggest a three-miRNA upregulation profile of miR-320c, miR-200c-3p, and miR-449c-5p in BAL for the first time, which appears to predict the development of COPD in smokers. Consequently, the joint upregulation in miR-320c, miR-200c-3p, and miR-449c-5p levels could serve as potential BAL biomarker to better discriminate COPD from smoker and non-smoker subjects, but these results need to be validated in a new, independent cohort. Moreover, in silico analysis found that these three miRNAs are potentially involved in the regulation of EGFR and Hippo pathways, which are linked to COPD progression. Even though these findings need to be validated in a larger cohort, they are encouraging and open potential new avenues for the tailored treatment of COPD based on miRNA profiling.